The enormous impact of obesity and type 2 diabetes demands that we develop a better understanding of how nutritional signals are interpreted and integrated by metabolically active tissues. In particular, there is newfound appreciation that adipose tissue serves as an endocrine organ with responsibility for coordinating a wide range of homeostatic mechanisms. The first of the adipose-derived hormones (the so-called 'adipokines') to be discovered was leptin, encoded by the Lep gene in a more or less adipose-selective manner. Leptin is now known to serve an adipostatic role by regulating appetite, and also plays a major role in glucose homeostasis, immune function, reproduction, and bone density. Raising leptin levels is a useful therapeutic strategy in lipodystrophy and in some genetic forms of obesity, and may be helpful in a subset of common obesity. Despite these clinical implications and the enormous amount of basic scientific interest in leptin, virtually nothing is known about the transcriptional pathways that regulate leptin mRNA synthesis. Here we propose several innovative approaches to solving this longstanding problem. For example, we have used BAC transgenesis to perform gross mapping of the regions required for adipocyte-selective Lep expression with great success. The first Aim uses additional rounds of BAC transgenesis to further reduce the target area. The second Aim allows fine mapping of critical cis regions through high- throughput DNase hypersensitivity mapping, chromatin immunoprecipitation of modified histone, and electrophoretic mobility shift assays. The third Aim is directed at identifying trans-acting factors that affect Lep expression through a limited RNAi screen in Lep reporter cells. Taken together, these studies will provide enormous insight into the transcriptional basis of Lep adipocyte expression. Leptin is an adipocyte-derived hormone with profound effects on appetite and glucose tolerance, in addition to effects on other physiological processes including bone density, reproduction, and immune function. Despite a decade of intensive study, there are virtually no data on the critical transcriptional pathways that regulate leptin synthesis. This proposal uses a variety of innovative technologies to address this deficit, including BAC transgenesis, high-throughput DNase hypersensitivity analysis, and shRNA screening. [unreadable] [unreadable] [unreadable]